The long term objective of our research continues to be to understand the mechanisms by which extracellular signals are transduced into cellular responses in the heart. In this renewal the title of the grant has been changed to more specifically reflect the focus of this proposal. The central hypothesis is that membrane phospholipids, particularly phosphatidylinositides (PI) and phosphatidylcholine (PC) are hydrolyzed in response to hormones and generate products that serve as second messenger mediating cardiac responses. Studies will be carried out in primary cultures of cardiomyocytes prepared from embryonic chick, adult rat and neonatal rat heart. A number of cardioactive hormones, including muscarinic and alpha1-adrenergic receptor agonists, angiotensin II, thrombin and endothelin stimulate PI hydrolysis in these cells. These agents have not been directly shown to stimulate diacylglycerol (DAG) formation or to activate protein kinase C (PKC). Also unknown is whether these hormones induce PC hydrolysis. The specific aims of this proposal are to determine 1) whether agonists stimulate PC hydrolysis in myocytes, what phospholipases are activated, and by what molecular mechanisms (GTP- binding protein, activation through protein kinase) 2) if DAG is increased, if there is a significant basal or hormone stimulated level of ether-linked (particularly alkeny- acyl- or plasmalogenic) diglycerides, and if there is a significant contribution of PC to diglyceride formation 3) if the activation of PKC can be demonstrated by intact cell [3H]-PDB binding, by enzyme activation in permeabilized cells or by redistribution of immunoreactive PKC protein 4) the possible role of phospholipid generated second messengers in leading to c-fos induction and increases in specific gene expression in neonatal ventricular myocyte and in leading to ANF secretion in atrial myocytes. If cardioactive hormones stimulate the generation of phospholipid-derived second messengers that mediate ANF secretion or cause alterations in gene transcription like those seen in hypertrophy, the basic mechanisms leading to pathological changes in these functions may be clarified and may prove to be amenable to pharmacological treatment.